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Disialoganglioside GD2 is expressed by glioblastoma multiforme (GBM) cells representing a promising target for anti-GD2 immunotherapeutic approaches. The aim of the present study was to investigate anti-tumor efficacy of the chimeric anti-GD2 antibody (Ab) dinutuximab beta against GBM. Expression levels of GD2 and complement regulatory proteins (CRP; CD46, CD55 and CD59) on well-known and newly established primary tumor originated GBM cell lines were analyzed by flow cytometry. Ab-dependent cellular (ADCC) and complement-dependent cytotoxicity (CDC) mediated by dinutuximab beta against GBM cells were determined by a non-radioactive calcein-AM-based assay. Analysis of primary GBM cells revealed a heterogeneous GD2 expression that varied between the cell lines analyzed with higher expression levels in the tumor surface compared to the core originated cells. Both GD2-positive and -negative tumor cells were detected in every cell line analyzed. In contrast to CDC, ADCC mediated by dinutuximab beta was observed against the majority of GBM cells. Importantly, CDC-resistant cells showed high expression of the CRP CD46, CD55 and CD59. Our present data show anti-tumor effects mediated by dinutuximab beta against GBM cells providing a rationale for a GD2-directed immunotherapy against GBM. Due to high CRP expression, a combining of GD2-targeting with CRP blockade might be a further treatment option for GBM.

Neuroblastoma (NB) still remains a major challenge in pediatric oncology. We recently showed CD11b+-dependent upregulation of the PD-1/PD-L1 checkpoint on NB cells treated with the chimeric anti-GD2 antibody (Ab) ch14.18/CHO. Here, we report effects of reduction of CD11b+ myeloid suppressive cells on ch14.18/CHO immunotherapy against NB. Flow cytometry, immunohistochemistry and RT-PCR were used to assess tumor infiltrating leukocytes and expression of myeloid suppressive cell-associated genes. XTT assay was used to show impact of 5-FU on tumor and effector cells. Antitumor effects of the combined treatment with ch14.18/CHO and reduction of myeloid suppressive cells were evaluated in a syngeneic NB mouse model. Tumor tissue of untreated mice showed a strong infiltration by CD11b+ cells (53% of all tumor infiltrating leukocytes). RT-PCR analysis of tumors revealed strong expression of the myeloid suppressive cell-associated genes analyzed with the strongest induction of M-CSFr, CCL2, IL-1β, IL-4, IL-6 r, IL-8, Arg1, and NOS2. Compared to controls, application of anti-CD11b Ab resulted in reduction of both CD11b+ cells in tumors and expression of myeloid suppressive cell-associated genes as well as delayed tumor growth and prolonged survival. These effects could be further improved by 5-FU. Importantly, the combinatorial immunotherapy with ch14.18/CHO and 5-FU showed the strongest antitumor effects and superior survival rates. In conclusion, reduction of immune suppressive myeloid cells augments anti-NB efficacy of a ch14.18/CHO-based immunotherapy representing a new effective treatment strategy against GD2-positive cancers.

Immunotherapy with short term infusion (STI) of monoclonal anti-GD2 antibody (mAb) ch14.18 (4 × 25 mg/m2/d; 8–20 h) in combination with cytokines and 13-cis retinoic acid (RA) prolonged survival in...

The disialoganglioside GD2 is a well-established target antigen for passive immunotherapy in neuroblastoma (NB). Despite the recent success of passive immunotherapy with the anti-GD2 antibody ch14.18 and cytokines, treatment of high-risk NB remains challenging. We expanded the approach of GD2-specific, antibody-based immunotherapy to an application of a GD2-specific natural killer (NK) cell line, NK-92-scFv(ch14.18)-zeta. NK-92-scFv(ch14.18)-zeta is genetically engineered to express a GD2-specific chimeric antigen receptor generated from ch14.18. Here, we show that chimeric receptor expression enables NK-92-scFv(ch14.18)-zeta to effectively lyse GD2(+) NB cells also including partially or multidrug-resistant lines. Our data suggest that recognition of GD2 by the chimeric receptor is the primary mechanism involved in NK-92-scFv(ch14.18)-zeta-mediated lysis and is independent of activating NK cell receptor/ligand interactions. Furthermore, we demonstrate that NK-92-scFv(ch14.18)-zeta is able to mediate a significant anti-tumor response in vivo in a drug-resistant GD2(+) NB xenograft mouse model. NK-92-scFv(ch14.18)-zeta is an NB-specific NK cell line that has potential for future clinical development due to its high stability and activity toward GD2(+) NB cell lines.

Immunotherapy with anti-GD2 antibody (Ab) ch14.18/CHO is effective for treatment of high-risk neuroblastoma (NB) patients and is mainly based on GD2-specific Ab-dependent cellular cytotoxicity (ADCC). Strategies to further enhance the efficacy are important and currently explored in prospective clinical trials randomizing ch14.18/CHO ± IL-2. Recently, expression of programmed death 1 (PD-1) inhibitory receptor by effector cells and its ligand (PD-L1) by tumor cells has been shown. Here, we report for the first time effects of PD-1 blockade on ch14.18/CHO-based immunotherapy and mechanisms involved. Expression of PD-1 and PD-L1 on NB and effector cells was analyzed by RT-PCR and flow cytometry in the presence of ch14.18/CHO and/or IL-2. The effect of PD-1 blockade on ch14.18/CHO-mediated anti-NB immune response was evaluated using anti-PD-1 Ab both in vitro (Nivolumab) and in a syngeneic PD-L1+/GD2+ NB mouse model (anti-mouse PD-1). Culture of NB cells LA-N-1 (low PD-L1 baseline expression) with leukocytes and subtherapeutic ch14.18/CHO concentrations for 24 h induced strong upregulation of PD-L1, which was further increased by IL-2 resulting in complete inhibition of ch14.18/CHO-mediated ADCC. Importantly, blockade with Nivolumab reversed the PD-L1-dependent inhibition of ADCC. Similarly, co-incubation with anti-CD11b Ab abrogated the PD-L1 upregulation and restored ADCC. Mice treated with ch14.18/CHO in combination with PD-1 blockade showed a strong reduction of tumor growth, prolonged survival and the highest cytotoxicity against NB cells. In conclusion, ch14.18/CHO-mediated effects upregulate the inhibitory immune checkpoint PD-1/PD-L1, and combination of ch14.18/CHO with PD-1 blockade results in synergistic treatment effects in mice representing a new effective treatment strategy against GD2-positive cancers.

Polymorphisms in Fc-gamma-receptor (FCGR) genes as well as killer cell immunoglobulin-like receptor (KIR) and KIR ligand (KIRL) repertoires may influence antitumor effects of monoclonal antibodies (mAb). Here, we systematically analyzed high- and low-affinity FCGR2A and -3A genotypes as well as stimulating and inhibitory KIR/KIRL combinations in 53 neuroblastoma (NB) patients treated by long-term infusion (LTI) of anti-GD2 IgG1 Ab ch14.18/CHO using validated real-time PCR methods. Patients with high-affinity FCGR2A and -3A genotypes showed a higher level of Ab-dependent cell-mediated cytotoxicity (ADCC) on day 8 after the start of ch14.18/CHO and superior event-free survival (EFS) compared to patients with low FCGR genotypes. Similar observations were made for patients with stimulatory KIR/KIRL haplotype B (combination of KIR genes including activating receptor genes) compared to inhibitory haplotype A (a fixed set of genes encoding for inhibitory receptors, except 2DS4) and stronger effects were found in patients when haplotype B and high-affinity FCGRs were combined. Surprisingly, independent analysis of KIRs showed a major role of activating KIR 2DS2 for high ADCC levels and prolongation of EFS. The greatest effect was observed in 2DS2-positive patients that also had high-affinity FCGR2A and -3A genotypes. In summary, the presence of the activating KIR 2DS2 has a major effect on ADCC levels and survival in NB patients treated by LTI of ch14.18/CHO and may therefore be a useful biomarker in combination with FCGR polymorphisms for Ab-based immunotherapies.

Ch14.18 manufactured in Chinese hamster ovary (CHO) cells is currently being evaluated in clinical trials. Short-term infusion (STI) (8-20 h/day; 4-5 days) of 100 mg/m2 ch14.18/CHO (dinutiximab β) per cycle in combination with cytokines is standard treatment of neuroblastoma (NB) patients. As pain is a limiting factor, we investigated a novel delivery method by continuous long-term infusion (LTI) of 100 mg/m2 over 10 days. 53 NB patients were treated with 5-6 cycles of 6 × 106 IU/m2 subcutaneous interleukin-2 (d 1-5, 8-12), LTI of 100 mg/m2 ch14.18/CHO (d 8-18) and 160 mg/m2 oral 13-cis-retinoic acid (d 22-35). Human anti-chimeric antibody (HACA), antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity were determined. With LTI, we observed a maximum concentration of ch14.18/CHO (Cmax) of 12.56 ± 0.68 µg/ml and a terminal half-life time (t1/2 β) of 32.7 ± 16.2 d. The clearance values for LTI and STI of 0.54 ± 0.13 and 0.41 ± 0.29 L/d m2 and area under the serum concentration-time curve (AUC) values of 189.6 ± 41.4 and 284.8 ± 156.8 µg×d/ml, respectively, were not significantly different. Importantly, we detected ch14.18/CHO trough concentration of ≥ 1 µg/ml at time points preceding subsequent antibody infusions after cycle 1, allowing a persistent activation of antibody effector mechanisms over the entire treatment period of 6 months. HACA responses were observed in 10/53 (19%) patients, similar to STI (21%), indicating LTI had no effect on the immunogenicity of ch14.18/CHO. In conclusion, LTI of ch14.18/CHO induced effector mechanisms over the entire treatment period, and may therefore emerge as the preferred delivery method of anti-GD2 immunotherapy to NB patients.

10530Background: Long-term infusion (LTI) of the anti-GD2 antibody (Ab) ch14.18/CHO in combination with interleukin-2 (IL-2) prolongs survival in patients (pts) with high-risk neuroblastoma compare...

Background The monoclonal anti-GD2 antibody ch14.18/CHO in combination with IL-2 is active and effective in high-risk neuroblastoma (NB) patients. Here, we investigated the inflammatory response and treatment tolerance of long-term infusion (LTI) of ch14.18/CHO (10 × 10 mg/m2 ; 24 hr) in combination with subcutaneous (s.c.) IL-2 in a single center program. Methods Fifty-three NB patients received up to six cycles of 100 mg/m2 ch14.18/CHO (d8-18, where d represents day(s)) as LTI combined with 6 × 106 IU/m2 s.c. IL-2 (d1-5; 8-12) and 160 mg/m2 oral 13-cis retinoic acid (RA) (d19-32). Side effects of ch14.18/CHO and IL-2 treatment require hospitalization of patients on d8. Treatment tolerance was evaluated daily with clinical parameters (body temperature, vital signs, Lansky performance status, requirement of i.v. concomitant medication) to define an outpatient candidate status. sIL-2-R and C-reactive protein values were determined to assess the inflammatory response. Results LTI of ch14.18/CHO (d8-18) in combination with s.c.IL-2 (d8-12) showed an acceptable treatment tolerance that allowed all patients to receive part of the treatment as an outpatient (median time point of discharge: d15 for all cycles). The treatment tolerance improved from cycle to cycle and the time to become an outpatient candidate decreased from d15 to d13 in subsequent cycles. Clinical and laboratory parameters indicate a maximum inflammatory response at d11 of each cycle. Interestingly, the soluble IL-2 receptor remained increased at baseline of the next cycle indicating immune activation over the entire treatment period of 6 months. Conclusions LTI of ch14.18/CHO combined with s.c.IL-2 shows an improved tolerance in subsequent cycles allowing outpatient treatment.

10548 Background: Immunotherapy (IT) with anti-GD2 antibody (Ab) ch14.18/CHO is effective for treatment of high-risk neuroblastoma (NB) patients mainly due to induction of GD2-specific Ab-dependent cellular cytotoxicity (ADCC). Methods to further enhance the effect are important and currently explored in prospective clinical trials randomizing ch14.18/CHO ± scIL-2. Programmed death-1 (PD-1) is an inhibitory receptor expressed by activated T- and NK-cells, and cancer cells express PD-1 ligand. Here, we report for the first time effect and mechanism of PD-1/PD-L1 blockade in the context of ch14.18/CHO-based IT in preclinical models. Methods: Expression of PD-L1 and PD-1 on NB cells and leukocytes was analyzed by RT-PCR and flow cytometry in the presence of ch14.18/CHO or IL-2. Mechanism of PD-L1 induction was analyzed with anti-CD11b Ab. The effect of PD-1/PD-L1 blockade on ch14.18/CHO-mediated anti-NB immune response was evaluated using anti-PD-1 Ab both in vitro (Nivolumab) and in the syngeneic GD2+ NB NXS2 mouse model (anti-mouse PD-1). Mice (n = 10) were treated with ch14.18/CHO (5x300 µg, i.p.) in combination with anti-PD-1 (8x250 µg, i.p.), and compared to controls. Results: Culture of LA-N-1 (low PD-L1 baseline expression) in the presence of leukocytes and subtherapeutic ch14.18/CHO concentrations for 24h induced strong upregulation of PD-L1 resulting in complete inhibition of ADCC mediated by ch14.18/CHO. Co-incubation with anti-CD11b Ab abrogated this PD-L1 upregulation. Importantly, blockade with Nivolumab reversed the PD-L1-dependent inhibition of ADCC. Mice treated with ch14.18/CHO in combination with PD-1 blockade showed a strong reduction of tumor growth and prolonged survival as well as the highest level of NB cell lysis mediated by serum and leukocytes compared to controls. Conclusions: Ch14.18/CHO-mediated effects upregulate the inhibitory checkpoint PD-1/PD-L1 and combination of ch14.18/CHO with PD-1/PD-L1 blockade results in synergistic treatment effects in mice. This concept will be evaluated in a clinical trial.

Human/mouse chimeric monoclonal antibody (mAb) ch14.18/CHO is directed against disialoganglioside GD2. Activity and efficacy of this mAb are currently determined in ongoing clinical Phase II and -III studies in high-risk neuroblastoma (NB). Based on the chimeric nature of this mAb, some patients may develop a human anti-chimeric immune response (Mirick et al., 2004) which impacts on pharmacokinetics and may induce anti-anti-idiotype (Id) mAb with a potential survival benefit. Therefore, a validated method of quantitative detection of human anti-chimeric antibodies (HACA) in serum samples of NB patients treated with ch14.18/CHO is an important tool for monitoring of clinical trials. Here, we report a validated sandwich enzyme-linked immunosorbent assay (ELISA) according to the one arm binding principle using ch14.18/CHO as a capture mAb and biotinylated ch14.18/CHO mAb for detection. Ganglidiomab, a monoclonal anti-Id Ab to ch14.18/CHO (Lode et al., 2013), was used as a standard for assay validation and HACA quantification. Systematic evaluation of the established ELISA procedure revealed an optimal serum sample dilution factor of 1:160. Assay validation was accomplished with a set of tailored quality controls (QC) containing distinct concentrations of ganglidiomab (3 and 15μg/ml). The coefficients of variation (CV) for all within-assay and inter-assay measurements using QCs were under 20% and the limit of detection (LOD) was 1.1μg/ml. Three patients (P1, P2, P3) treated with a 10day continuous infusion of 100mg/m(2) of ch14.18/CHO were selected for analysis with this assay. Selection was based on ch14.18/CHO drug level on day 8 in cycle 2 of >10μg/ml (expected) (P1) and of

Human/mouse chimeric monoclonal antibody (mAb) ch14.18 is directed against disialoganglioside GD2 and has demonstrated activity and efficacy in high-risk neuroblastoma (NB). For the purpose of industrial production, ch14.18 was manufactured in Chinese hamster ovarian cells (ch14.18/CHO) in order to facilitate clinical trials in Europe. To determine immunopharmacological effects of ch14.18 in preclinical models and clinical trials, a validated method of quantitative detection of ch14.18/CHO in serum is an important tool. We recently described the generation and characterization of ganglidiomab, a monoclonal anti-idiotype Ab (AIT) of ch14.18 (Lode et al., 2013), which was used to establish quantitative and validated enzyme-linked immunosorbent assay (ELISA) methods using ganglidiomab as a capture mAb. With these ELISA methods, we first demonstrated binding of ch14.18/CHO to ganglidiomab to a similar extent as to the nominal antigen GD2 and in contrast to GD1b and GM2 precursor and metabolite gangliosides, used as negative controls. In order to determine both low (0.5-3.1 μg/ml) and high levels of ch14.18/CHO (1.0-25 μg/ml) in the serum of NB patients treated with ch14.18/CHO, we established two ELISA methods with high and low sensitivity using 1/1001, and 1/5126 sample dilutions, respectively. For validation, we used a set of tailored quality controls (QC) containing distinct concentrations of ch14.18/CHO (1.0, 2.0, 7.0, and 20.0 μg/ml). We determined the limit of detection (LOD) for both ELISA methods to be 0.50 μg/ml for the high sensitivity and 1.02 μg/ml for low sensitivity ELISA. The within-assay precision was 12% for high and 4% for low sensitivity ELISA, and the coefficients of variation (CV) were under 20% for all assays (3% for QC-1.0, 5% for QC-2.0, 7% for QC-7, and 3% for QC-20). With this method, we showed that neither eight freeze-thaw cycles nor storage at room temperature for up to 168 h affected ch14.18/CHO stability in serum. Finally, we analyzed ch14.18 Ab serum levels in selected NB patients receiving ch14.18/CHO as a continuous or bolus infusion with a peak concentration at the last day of Ab application (17.14 ± 7.20mg/ml with continuous and 19.78 ± 2.26 mg/ml with bolus infusion). In summary, we describe validated ELISA methods using ganglidiomab as a capture mAb suitable for the pharmacological evaluation of ch14.18/CHO in NB patients.

10562Background: A new delivery method of anti-GD2 antibody ch14.18/CHO by long term infusion (LTI) may improve the toxicity profile but maintain effective immune modulation and clinical activity i...

10563Background: Long-term infusion (LTI) emerges as a promising new delivery method of ch14.18/CHO for relapsed/refractory neuroblastoma patients. The role of concomitant treatment with scIL2 is u...

Vaccination with proteins mimicking GD2 that is highly expressed on neuroblastoma (NB) cells is a promising strategy in treatment of NB, a pediatric malignancy with poor prognosis. We previously showed efficacy of ganglidiomab in vivo, a murine anti-idiotype (anti-Id) IgG1. In order to tailor immune responses to variable regions, we generated a new human/mouse chimeric anti-Id antibody (Ab) ganglidiximab by replacing murine constant fragments with corresponding human IgG1 regions. DNA sequences encoding for variable regions of heavy (VH) and light chains (VL) were synthesized by RT-PCR from total RNA of ganglidiomab-producing hybridoma cells and further ligated into mammalian expression plasmids with coding sequences for constant regions of human IgG1 heavy and light chains, respectively. We established a stable production cell line using Chinese hamster ovarian (CHO) cells co-transfected with two expression plasmids driving the expression of either ganglidiximab heavy or light chain. After purification from supernatants, anti-idiotypic characteristics of ganglidiximab were demonstrated. Binding of ganglidiximab to anti-GD2 Abs of the 14.18 family as well as to NK-92tr cells expressing a GD2-specific chimeric antigen receptor (scFv(ch14.18)-zeta) was shown using standard ELISA and flow cytometry analysis, respectively. Ganglidiximab binding affinities to anti-GD2 Abs were further determined by surface plasmon resonance technique. Moreover, binding of anti-GD2 Abs to the nominal antigen GD2 as well as GD2-specific Ab-mediated cytotoxicity (ADCC, CDC) was competitively inhibited by ganglidiximab. Finally, ganglidiximab was successfully used as a protein vaccine in vivo to induce a GD2-specific humoral immune response. In summary, we report generation and characterization of a new human/mouse chimeric anti-Id Ab ganglidiximab for active immunotherapy against NB. This Ab may be useful to tailor immune responses to the paratope regions mimicking GD2 overexpressed in NB.

Immunotherapy targeting disialoganglioside GD(2) emerges as an important treatment option for neuroblastoma, a pediatric malignancy characterized by poor outcome. Here, we report the induction of a GD(2)-specific immune response with ganglidiomab, a new anti-idiotype antibody to anti-GD(2) antibodies of the 14.18 family.Ganglidiomab was generated following immunization of Balb/c mice with 14G2a, and splenocytes were harvested to generate hybridoma cells. Clones were screened by ELISA for mouse antibody binding to hu14.18. One positive clone was selected to purify and characterize the secreted IgG protein (κ, IgG(1)). This antibody bound to anti-GD(2) antibodies 14G2a, ch14.18/CHO, hu14.18, and to immunocytokines ch14.18-IL2 and hu14.18-IL2 as well as to NK-92 cells expressing scFv(ch14.18)-zeta receptor. Binding of these anti-GD(2) antibodies to the nominal antigen GD(2) as well as GD(2)-specific lysis of neuroblastoma cells by NK-92-scFv(ch14.18)-zeta cells was competitively inhibited by ganglidiomab, proving GD(2) surrogate function and anti-idiotype characteristics. The dissociation constants of ganglidiomab from anti-GD(2) antibodies ranged from 10.8 ± 5.01 to 53.5 ± 1.92 nM as determined by Biacore analyses. The sequences of framework and complementarity-determining regions of ganglidiomab were identified. Finally, we demonstrated induction of a GD(2)-specific humoral immune response after vaccination of mice with ganglidiomab effective in mediating GD(2)-specific killing of neuroblastoma cells.We generated and characterized a novel anti-idiotype antibody ganglidiomab and demonstrated activity against neuroblastoma.

Cytotoxic T lymphocytes and their granule components, such as perforin and granzyme, play an important role in the defense of hepatic infections caused by different pathogens. Moreover, it has been shown in vitro that hepatocytes can initiate cell death via a perforin-dependent mechanism. Although it is well known that hepatocellular apoptosis in D-galactosamine/lipopolysaccharide (D-Gal/LPS)-associated liver failure is mediated by TNF-alpha-dependent Fas/FasL cytotoxicity, there is no information on the role of perforin-mediated mechanisms in vivo. Therefore, we studied whether the cytolytic perforin/granzyme pathway contributes to the D-Gal/LPS-associated hepatotoxicity. Perforin knockout (Pko) mice showed significantly higher hepatic TNF-alpha and IL-6 mRNA expression as well as plasma TNF-alpha and IL-6 concentrations within the first hour upon D-Gal/LPS challenge compared with perforin wild-type (Pwt) mice. At 6 h upon D-Gal/LPS challenge, Pko mice further presented with higher transaminase release and onconecrotic tissue damage, whereas hepatocellular apoptosis and caspase-3 cleavage remained unaffected by the perforin deficiency. Pretreatment with a recombinant human TNF-alpha receptor fusion protein attenuated necrotic and apoptotic tissue damage and reduced plasma transaminase activities as well as cytokine release, thereby preventing acute liver failure in Pko mice as effectively as in Pwt mice. These data do not only confirm the significance of TNF-alpha as distal mediator of hepatic injury in this model but simultaneously reveal a contribution of a perforin-dependent immunoregulation, limiting the D-Gal/LPS-induced overwhelming cytokine release and onconecrotic tissue injury.

There is increasing evidence that the active contribution of hepatocytes to liver disease is strongly dependent on local cytokine environment. It has been shown in vitro that TNFalpha can enhance hepatocyte FasLigand (FasL)-mediated cytotoxicity. Here, we demonstrate that TNFalpha-induced apoptosis was associated with Fas and FasL upregulation and that a FasL-neutralizing antibody prevented TNFalpha-induced apoptosis. We further examined in vivo the relevance of the Fas/FasL pathway to hepatocellular apoptosis in a TNFalpha-driven model of acute liver failure. Livers of galactosamine/lipopolysaccharide (Gal/LPS)-exposed Fas wild-type mice highly expressed both Fas and FasL and revealed marked hepatocellular apoptosis that was almost completely blocked by soluble TNFalpha-receptor; this was also almost absent in Gal/LPS-exposed Fas lymphoproliferation mutant mice. Our data provide evidence for a direct link between TNFalpha and Fas/FasL in mediating hepatocyte apoptosis. Fratricidal death by Fas-FasL interactions of neighbouring hepatocytes may actively contribute to acute liver failure.

3029 Background: Neuroblastoma stage IV is associated with low cure rates. Treatment with the GD2 antibody ch14.18 could improve the long-term overall survival. Apart from GD2 other tumor antigens (TA) are expressed in neuroblastoma and studies implicated that T-cell responses to TA are associated with a better outcome. For the present study we analyzed the immune response in long term neuroblastoma survivors treated with anti GD2 ch14.18. Methods: Blood and serum of 17 neuroblastoma long-term survivors after GD2 therapy with > 5 years follow up (patients group) and 17 age and gender matched healthy donors (control group) were analyzed. Serum was analyzed for antibodies binding to neuroblastoma cell lines and antibodies to a nominal antigen GD2. T cells were stimulated with overlapping peptides of the tumor antigens (TA) NY-ESO-1, WT-1, Survivin, MAGE-A1 and two mimotope peptides to GD2 for 10 days and analyzed for IFN-γ secretion in flow cytometry. Results: T-cell responses to at least one TA could be fo...

TPS10080 Background: Long-term infusion (LTI) of anti-GD2 antibody ch14.18/CHO may improve outcome in patients (pts) with high risk relapsed/refractory neuroblastoma (NB). Methods: 97 pts received ...

Purpose: Immunotherapy with monoclonal anti-GD2 antibody (mAb) ch14.18 in combination with cytokines effectively prolonged survival in high risk neuroblastoma (NB). We piloted a less toxic treatment using continuous infusion of ch14.18/CHO in combination with subcutaneous interleukin-2 (IL-2) and report initial clinical response and survival. Methods and Materials: 53 high risk NB patients received up to 6 cycles of 6x106 IU/m2 s.c. IL-2 (d1-5; 8-12), continuous infusion of 100 mg/m2 ch14.18/CHO (d8-17) and 160 mg/m2 oral 13-cis-RA (d19-32) in a single center compassionate use program. Killer immunoglobulin-like receptor (KIR)/KIR-ligand (KIRL) mismatch and Fcγ-receptor (FCGR) was analyzed with a validated PCR-based analysis of KIR, HLA, FCGR2A (H131R), -3A (V158F) and -3B (NA1/NA2) polymorphisms. Antibody levels and GD2 specific killing of neurpblastoma cells by ADCC, CDC, and whole blood were sequentially analyzed. Clinical response was assessed by mIBG, MRI/CT, bone marrow- and catecholamine- analysis before, after 2/3 and after 5/6 cycles of immunotherapy in patients with measurable disease. Results: : In the patients receiving ch14.18/CHO continuous infusions significantly less i.v. morphine was used compared to patients who receive ch14.18/CHO bolus infusions. In many patients it was possible to discontinue i.v. morphine and to treat pain with oral Gabapentin only. Response rates were 41.7 % in mIBG (15/36), 31.8 % MRI/CT (7/22), 28.6 % bone marrow- (6/21) and 38.1 % in catecholamine- (8/21) measurements. External review of mIBG responses of 28 patients confirmed a 32.1% response rate (9/28). The overall response following INRG guide lines was determined at 30% (12/40). The event-free-survival rate in the entire cohort was 32.4 % (observation 3.2 years, mean: 1.6 years) and an overall-survival rate of 66.8% (observation 3.9 years, mean: 3.1 years). Patients with KIR/KIRL mismatch and high affinity FCGR alleles showed a trend towards longer event-free survival (P = 0.08), which supports NK cell mediated antibody dependent cytotoxicity as the mechanism of action. Determination of antibody levels and functional immune parameters (ADCC, CDC and WBT) indicate persistent anti-neuroblastoma immune-activity measurable before susequent treatment cycles. Conclusion: Continuous infusion of anti-GD2 Ab ch14.18/CHO reduces the pain side effect, shows clinical activity in NB patients and improves the outcome. Analysis of KIR/KIRL-mismatch and FCGR-polymorphisms by genotyping may predict clinical outcome in patients receiving ch14.18/CHO. Functional immune parameters indicate anti-neuroblastoma activity over the entire treatment period. These results are subject to prospective confirmation in a SIOPEN Phase II study (EudraCT: 2009-018077-31). Citation Format: Holger Lode, Christian Jensen, Nikolai Siebert, Silke Kietz, Karoline Ehlert, Ina Müller, Ruth Ladenstein, Evelyne Janzek, Hans Loibner. Immune activation, clinical response and survival following long-term infusion of anti-GD2 antibody ch14.18/CHO in combination with interleukin-2 in high-risk neuroblastoma patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT410. doi:10.1158/1538-7445.AM2014-CT410

Effective treatment of high-risk neuroblastoma (NB) remains a major challenge in pediatric oncology. Human/mouse chimeric monoclonal anti-GD2 antibody (mAb) ch14.18 is emerging as a treatment option to improve outcome. After establishing a production process in Chinese hamster ovary (CHO) cells, ch14.18/CHO was made available in Europe for clinical trials. Here, we describe validated functional bioassays for the purpose of immune monitoring of these trials and demonstrate GD2-specific immune effector functions of ch14.18/CHO in treated patients. Two calcein-based bioassays for complement-dependent- (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were set up based on patient serum and immune cells tested against NB cells. For this purpose, we identified LA-N-1 NB cells as best suited within a panel of cell lines. Assay conditions were first established using serum and cells of healthy donors. We found an effector-to-target (E:T) cell ratio of 20:1 for PBMC preparations as best suited for GD2-specific ADCC analysis. A simplified method of effector cell preparation by lysis of erythrocytes was evaluated revealing equivalent results at an E:T ratio of 40:1. Optimal results for CDC were found with a serum dilution at 1:8. For validation, both within-assay and inter-assay precision were determined and coefficients of variation (CV) were below 20%. Sample quality following storage at room temperature (RT) showed that sodium-heparin-anticoagulated blood and serum are stable for 48 h and 96 h, respectively. Application of these bioassays to blood samples of three selected high-risk NB patients treated with ch14.18/CHO (100 mg/m(2)) revealed GD2-specific increases in CDC (4.5-9.4 fold) and ADCC (4.6-6.0 fold) on day 8 compared to baseline, indicating assay applicability for the monitoring of multicenter clinical trials requiring sample shipment at RT for central lab analysis.

Immunotherapy of neuroblastoma with anti-GD2 antibodies in combination with interleukin-2 (IL2) emerges as an important treatment option as shown in several clinical trials. In consequence, an antibody-IL2 fusion protein (hu14.18-IL2; immunocytokine) was designed that combines the targeting and effector functions of an anti-GD2 antibody and the immune modulation activity of IL2. In addition to mediation of typical effector functions such as CDC and ADCC this immunocytokine targets IL2 to GD2+ tumor cells and thereby links tumor cells to IL2-receptor bearing effector cells via an Fc-gamma independent mechanism. This concept has already shown encouraging results in Phase I/II trials in relapsed/refractory neuroblastoma patients. Here, we report the antigen binding characteristics and in vitro anti-neuroblastoma activity of a new GMP preparation of hu14.18-IL2 (APN301). Binding of APN301 to its nominal antigen GD2 was analyzed by solid phase ELISA and compared to other antibodies of the 14G2a family. Affinities of APN301 to GD2 and to anti-14.18 anti-idiotype antibodies were also determined by Biacore® analysis. GD2 specific ADCC, CDC and whole blood cytotoxicity were analyzed in cytotoxicity assays in vitro (calcein-release, 51Cr release) using GD2+ neuroblastoma target cell lines. We demonstrate similar binding characteristics of APN301 to GD2 compared to parent 14.18 antibodies (murine and chimeric). The anti-14.18 anti-id antibody ganglidiomab fully inhibits binding of APN301 to the nominal antigen GD2, thereby proving internal image properties. The dissociation constants of APN301 to GD2 and ganglidiomab as determined in Biacore analyses are in the 10-9M and 10-7M range, respectively, similar to those determined for the naked chimeric 14.18 antibody. Relevant for the prediction of clinical activity we demonstrate potent specific lysis of GD2+ human neuroblastoma cells mediated by APN301 in a whole blood cytotoxicity assay in vitro, utilizing whole blood of healthy donors as well as whole blood of high-risk neuroblastoma patients in advanced disease stage. Further clinical trials with APN301 in neuroblastoma patients are being planned, taking into account the found potent whole blood cytolytic activity and the pharmakokinetic properties of this immunocytokine. Citation Format: Hans Loibner, Manfred Schuster, Evelyne Janzek, Stefan Stranner, Bernhard Peball, Susanne Wiederkum, Oliver Mutschlechner, Nikolai Siebert, Holger Lode. Binding characteristics of the immunocytokine hu14.18-IL2 and induction of human effector functions as anticipated mode of action. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2858. doi:10.1158/1538-7445.AM2013-2858 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Purpose: Immunotherapy targeting disialoganglioside GD2 emerges as an important treatment option for neuroblastoma, a pediatric malignancy characterized by poor outcome. Here, we report the generation and characterization of ganglidiomab, a new anti-idiotype antibody to anti-GD2 antibodies of the 14.18 family for monitoring of clinical trials and the development of neuroblastoma vaccines. Experimental Design and Results: Balb/c mice were immunized with 14G2a and splenocytes harvested to generate hybridoma cells. Clones were screened by ELISA for mouse antibody binding to hu14.18. One positive clone was selected to purify and characterize the secreted IgG protein (κ, IgG1). This antibody bound to anti-GD2 antibodies 14G2a, ch14.18/CHO, hu14.18 and to immunocytokines ch14.18-IL2 and hu14.18-IL2 as well as to NK-92 cells expressing scFv(ch14.18)-zeta receptor. Binding of these anti-GD2 antibodies to the nominal antigen GD2 as well as GD2 specific lysis of neuroblastoma cells by NK-92-scFv(ch14.18)-zeta cells was competitively inhibited by ganglidiomab, proving GD2 surrogate function and anti-idiotype characteristics. The dissociation constants of ganglidiomab from anti-GD2 antibodies ranged from 10.8 ± 5.01 to 53.5 ± 1.92 nM as determined by Biacore analyses using “steady state” analysis. The sequences of framework- (FRs) and complementarity determining -regions (CDRs) of ganglidiomab were identified. Finally, we demonstrate induction of a GD2 specific humoral immune response after vaccination of mice with ganglidiomab effective in mediating GD2 specific killing of neuroblastoma cells. Conclusion: We generated and characterized a novel anti-idiotype antibody ganglidiomab for immune monitoring of clinical trials with anti-GD2 antibodies and provide an important baseline for the development of anti-idiotype vaccines against malignancies expressing GD2. Citation Format: Holger N. Lode, Manuela Schmidt, Diana Seidel, Nicole Huebener, Diana Brackrock, Matthias Bleeke, Daniel Reker, Sven Brandt, Hans-Peter Mueller, Christiane Helm, Nikolai Siebert. Generation and characterization of anti-idiotype antibody ganglidiomab as GD2 surrogate for immunotherapy of neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A28.

Meeting abstracts GD2 is highly expressed in neuroblastoma (NB) and specifically recognized by antibodies ch14.18. Immunotherapy with bolus infusion of ch14.18 in combination with cytokines prolonged survival in patients at risk for relapse but was associated with substantial toxicity, in